Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the oldest pathogens that have impacted humans globally and the re-emergence of M. tuberculosis has become a primary public health burden (National Institute of Allergy and Infectious Diseases, Tuberculosis Health Matters 2002. Fact sheets and brochure; Raviglione, M. C., 2003, Tuberculosis 83:4-14; World Health Organization. 2005. WHO declares TB an emergency in Africa: Call for “urgent and extraordinary actions” to halt worsening epidemic. WHO Media centre News). The rise in multi-drug resistant and extensively drug-resistant strains of M. tuberculosis has reduced the impact of current treatment options (Cole, S. T. et al. 1998. Nature 393:537-544; Fauci, A. S., 2008. Perspective JID 197:1493-1498; Zhang, Y. 2005. Annu. Rev. Pharmacol. Toxicol. 45:529-564). Therefore, the development of antibiotics with novel mechanisms of action that will be effective for short term therapy is essential to effectively treat patients with tuberculosis (TB).
Methionine Aminopeptidase (MetAP) is a dinuclear metalloprotease that removes the N-terminal methionine from polypeptides and proteins (Giglione, C., A. Boularot, and T. Meinnel. 2004. Cell. Mol. Life. Sci. 61:1455-1474; Lowther, W. T., and B. W. Matthews. 2000. Bioch Biophys. Acta. 1477:157-167). MetAP is conserved in all microbial genomes that have been sequenced to date. There are two classes of MetAPs, MetAP1 and MetAP2 that differ in the presence of an internal polypeptide insertion present within the catalytic domain of MetAP2 (Addlagatta, A., et al. 2005. Biochemistry 44:14741-14749; Arfin, S. M., et al. 1995. Proc. Natl. Acad. Sci. 92:7714-7718; Liu, S., et al. 1998. Science 282:1324-1327). Eukaryotes possess both classes while prokaryotes have homologs of either MetAP1 (eubacteria) or MetAP2 (archeabacteria) (Lowther, W. T., and B. W. Matthews. 2000. Biochim. Biophys. Acta. 1477:157-167). Variants of MetAP1 are further classified as MetAP1a, MetAP1b and MetAP1c (Addlagatta, et al. 2005. Biochemistry 44:7166-7174), which are distinguished by the existence of an N-terminal extension in MetAP1b and MetAP1c, and a unique zinc finger domain in MetAP1b. Recently, the X-ray crystal structures of the apo- and methionine-bound forms of M. tuberculosis MetAP1c were solved (Addlagatta, et al. 2005. Biochemistry 44:7166-7174). The X-ray structure revealed the existence of a highly conserved proline rich N-terminal extension in MtMetAP1c which is absent in MtMetAP1a but has sequence homology with the linker region of human MetAP1 (HsMetAP1).
Genetic studies have shown that deletion of MetAP from Escherichia coli and Salmonella typhimurium is lethal (Chang, S. Y., E. C. McGary, and S. Chang. 1989. J. Bacteriol. 171:4071-4072; Miller, C. G., J. L. Kukral, and N. R. Movva. 1989. J. Bacteriol. 171:5215-5217). In yeast, deletion of either ScMetAP1 or ScMetAP2 results in a slow-growth phenotype, while disruption of both genes is lethal. In Caenorhabditis elegans, MetAP2 is essential for germ cell development. In mammalian cells both HsMetAP1 and HsMetAP2 have been shown to be required for cell proliferation. Moreover, HsMetAP2 is essential for endothelial cell growth and angiogenesis. Recent studies from our lab have also shown that HsMetAP1 is involved in G(2)/M phase of the cell cycle.
The essential role of MetAPs in prokaryotes makes this enzyme an attractive target for the development of new antibiotics. In prokaryotes, where protein synthesis begins with an N-formylated methionine, peptide deformylase (PDF) catalyzes the removal of the formyl group before MetAP activity (Giglione, C., et al. 2003. EMBO J. 22:13-23; Solbiati, J., et al. 1999. J. Mol. Biol. 290:607-614). Unlike most other prokaryotes, M. tuberculosis possesses two MetAPs: MtMetAP1a and MtMetAP1c. They share about 33% sequence identity. Both MtMetAPs have less than 45% similarity to E. coli MetAP1 (EcMetAP1), less than 48% similarity to human MetAP1 (hMetAP1) and less than 30% similarity to human MetAP2 (hMetAP2). Given the presence of the two MetAP genes in M. tuberculosis, it was unclear whether inhibition of either or both MtMetAPs is sufficient to inhibit bacterial growth.